We have identified a candidate molecular chaperone (IP9O) that we hypothesize plays an important role in assembly of MHC class I complexes. mAb Af8 was made against the IP9O protein and revealed it to be an abundant protein resident in the ER. Using this mAb the gene encoding IP9O was cloned from a lambda-gt11 expression library. The gene is predicted to encode a transmembrane protein with a ER luminal domain of 461 aa, a transmembrane segment of 22 aa and a cytosolic COOH terminal tail of 89 aa. Pulse chase experiments reveal that IP9O associates with assembling polypeptides in the ER particularly at very short time periods following their biosynthesis. However, this association is transient and the recently synthesized polypeptides dissociate from IP9O in a time dependent fashion. In the case of MHC class I assembly, the free MHC class I heavy chain remains persistently associated with IP9O under conditions where assembly cannot be completed either because beta2 microglobulin or peptide antigen is lacking. Thus, IP9O may play a role in facilitating the assembly of the class I complex and/or mediate retention of the empty or incompletely assembled heavy chains in the ER. In order to characterize the function of IP9O and in particular its role in MHC class 1 assembly the following issues will be addressed. (1) We will determine if IP9O selectively binds transmembrane proteins by comparing association of IP9O with transmembrane vs alternatively spliced secreted MHC class I molecules. (2) Engineered IP9O molecules that leave the ER and move to the gold or the cell surface will be used to determine whether MHC class 1 free heavy chains exit the ER along with the mutant IP9O molecules, in order to assess its role in ER retention. (3) We will determine the domains of IP9O that interact with respective domains of MHC class 1 heavy chains by generating deletion and swap mutants of both molecules and examining the effects on association. (4) We will determine directly if IP9O facilitates assembly of class 1 heavy chain - beta2m- peptide complexes through in vitro reconstitution experiments in lysates depleted or reconstituted with IP9O protein. (5) We will examine the effect of IP9O depletion in vivo using antisense inhibition of IP9O gene expression in cultured lymphocytes. Together these experiments should elucidate important aspects of the biology of this candidate chaperone and determine its role in antigen presentation through mediating MHC class 1 assembly.